This application is a revision of our current funded project (parent grant) entitled Nanoassay for Realtime Molecular Probing of ABC Transporters (R01 GM076440, 05/01/2006-04/30/2011), submitted in response to Enabling RPGs to Leverage NCRR Center and Center-like Programs (NOT-OD-09-058) issued by NCRR, aiming to enhance the interaction of individual research programs with the NCRR center and enabling access to state-of-the-art instrumentation available at the center and thereby advance biomedical research. The justifications of this revision are given below: The study of membrane proteins at the molecular level is one of the major challenges for today's biologists. ATP-binding cassette (ABC) transporters are one of the largest and most diverse super families of membrane proteins found in living organisms ranging from bacteria to human. All ABC transporters share a common structural organization, suggesting a similar mechanism of energy coupling. ABC transporters are medically relevant. For instance, they are responsible for severe sicknesses and multidrug resistance (MDR) in bacterial infections and cancer chemotherapy. Despite extensive studies, the molecular mechanism of ABC transporters remains elusive and many questions remain unanswered. In our parent grant, we aim to develop photostable (non-photodecomposition and non-blinking) single nanoparticle optical assays, and to use them to mimic the substrates of the transporters, and as the probes to characterize the functional mechanism of BmrA, a multidrug bacterial transporter belonging to the ABC transporter super family. We have made significant progress, and have nearly completed Aims 1 and 2 in the parent grant, which is summarized in preliminary studies and our recent papers. In this revised application, we aim to expand our original proposed research scope to use high resolution cryo transmission-electron-microscopy (cryo-TEM), computer reconstruction and structure mining to solve the structure and molecular mechanism(s) of BmrA, a multidrug bacterial transporter belonging to the ABC transporter super family, by collaborating with the National Center for Macromolecular Imaging (NCMI) (a NCRR designated Biomedical Research Resource for Structural Biology, at http://ncmi.bcm.tmc.edu/). The specific research aims are described below: Aim 1: We will reconstitute purified BmrA into liposomes and use the single nanoparticle assay developed in our lab to determine their transport mechanism in real-time (ms-<s temporal resolution) using our single nanoparticle microscopy and spectroscopy, and use cryo-TEM at NCMI to determine the structure of BmrA and locations of single nanoparticles in BmrA at sub-nanometer (nm) resolution. We will compare the results acquired using cryo TEM with real-time data acquired using our single nanoparticle assay and optical microscopy. The combination of both approaches will enable us to use single nanoparticles as nm markers to trace the molecular mechanism of the ABC transporter, aiming to better understand how the ABC pump functions and its related molecular mechanisms. Aim 2: We have determined the transport kinetics of BmrA in single living cells in real-time using our single nanoparticle assay and single nanoparticle microscopy and spectroscopy. We will explore the possibility of using cryo-TEM to determine the structures of BmrA in cells (instead of liposomes) and the location of single nanoparticles in BmrAs in cells at sub-nm resolution, aiming to further develop cryo-TEM to be well suited to probe protein structures in its native environments (cellular membranes). The outcomes of the proposed research include: new nanoparticle assays for real-time measurement of membrane transport pathways and mechanisms of membrane transporters using optical microscopy, and as nm probes for tracing molecular mechanisms of membrane transporter using Cryo-TEM;new knowledge of transport mechanisms of nanoparticles in and out of living cells by ABC transporters;and new insights into the assembly and functioning of membrane proteins for better understanding of mechanisms of actions of ABC transporters, thus permitting more effective therapies. The proposed research will accelerate scientific research and offer new research and job opportunities, achieving the objectives of the Recovery Act. PUBLIC HEALTH RELEVANCE: The powerful imaging tools and novel assays described herein will offer new insights into the mechanisms and functions of ABC membrane transporters. Better understanding of the molecular mechanisms and functions of ABC transporters is essential step for one to develop more effective therapeutic treatments, notably for those transporters involved in multi-drug resistance (MDR) of bacteria and cancerous cells. Thus, the proposed research is health relevant. Furthermore, membrane transport plays a leading role in a wide spectrum of cellular and subcellular pathways, such as MDR, cellular signaling and cell-cell communication. Therefore, the proposed new nanoassay and imaging tools are expected to become extremely valuable tools to address numerous biochemical and biomedical problems associated with kinetics of membrane transports in living cells, as well as solving structure of membrane transporters at sub-nm resolution. Combination of real-time kinetic measurements with atomic scale structure characterization of membrane transporters will advance our understanding of membrane transporters and MDR.